The main objective of this Proposal is to identify one or more novel HIV-1 envelope (Env) proteins capable of inducing broadly neutralizing antibodies to numerous clinically relevant primary viral strains. This Proposal employs a powerful technology called directed molecular evolution. This approach involves the use of in vitro recombination to create a large number of chimeric genes encoding variant Env structures that will be systematically evaluated with respect to their antigenic and immunogenic properties. Our hypothesis is that some of these novel variant proteins will adopt conformations in which conserved neutralizing epitopes will induce broadly cross-reactive neutralizing antibodies. Two principal screening approaches are (i) the use of monoclonal antibodies that broadly neutralize HIV-1 and (ii) immunization studies in rabbits. In vitro recombination and the associated screening methods will first be applied to glycosylation variants of the gp140 Env protein. In a second step, amino acid diversity will be incorporated into the best glycosylation variant using consensus HIV sequences from Group M viruses. In a third step, directed molecular evolution will be used in a recursive manner to achieve further improvement in the immunogenicity of these Env variants. The best immunogens will also be tested in a soluble trimeric form by Project 2. Novel Env antigens that strongly bind the existing monoclonal antibodies that neutralize HIV-1 will be prepared for boosting the neutralizing immune response. In collaboration with Project 2, we will used directed molecular evolution to assist in the crystallization of the Env molecule and to select for virus-like particles that carry stable Env complexes on their surface. This Project will contribute to the overall goals of the HIVRAD Program in developing approaches to increase the immunogenicity of HIV antigens.